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Abstract

Optically guided neuron growth is a relatively new field where the exact mechanisms that initiate growth are not well understood. Both Gaussian light beams and optical line traps have been purported to initiate neuronal growth. Here we present a detailed study using optical line traps with symmetric and asymmetric intensity profiles which have been previously reported to bias the direction of neuronal growth. In contrast to these previous studies, we show similar levels of growth regardless of the direction of the intensity variation along the line trap. Furthermore, our experimental observations confirm previous suggestions that the filopodia produced from neuronal growth cones can be affected by laser light. We experimentally observe alignment of filopodia with the laser field and present a theoretical model describing the optical torques experienced by filopodia to explain this effect.

Figures (9)

The optical setup. A 1064 nm laser was focused at the sample plane after having its profile altered to be an asymmetric line. The cylindrical lens squeezes the beam in one dimension and the beam block truncates the profile to provide the asymmetry. It is important to note that the line formed by the cylindrical lens at the focus extends perpendicular to the plane of the diagram and so the beam block is raised slightly out of the plane so as not to block the whole beam. The profile of the laser is shown in the dotted circles before and after the cylindrical lens and beam block and it should be noted that since the line trap formed is perpendicular to the plane of the diagram, the asymmetrical profile displayed in the dotted circle is, so that it can be seen, not in the same orientation as the line trap. [Media 1]

Profile of beam taken with beam profiler. Left, oblique view of how the intensity profile of the asymmetric beam varies over space. Right, top down view of beam profile showing contour lines linking areas of same intensity.

A typical growth cone is shown above with an arrow added to represent an asymmetric line trap in the forward (right image) or reverse (left image) bias configuration. The dash in the arrow represents the most intense region of the beam. Actin would be expected to move along the arrow due to the ‘slingshot’ effect reported in [4]. On the left the laser is configured to induce an optical bias on the edge of the growth cone to grow along x (reverse bias) and on the right the laser is configured to produce the bias to grow along -x (forward bias). Scale bar is 10 µm.

Time lapsed sequence showing examples of neuronal growth in forward (left) and reverse bias (configurations). Frames flow from top to bottom; each frame is five minutes apart with time stamp in minutes in lower right corner. The field of view changes as the sample stage is moved to keep the beam on the growing cell. To compensate for this changing field of view a manually drawn outline is added to successive frames to represents where the edge of the growth cone was in the previous frame. Each neuron grows up until about frame 6 at which time they begin to stall and retract. Scale bar is 10 µm.

(439 KB) Movie of the alignment of filopodia on leading edge of a growth cone during exposure to a symmetric line trap. This particular example is representative of six observations of similar behavior, regardless of line trap bias. Speed is approximately 100 times and the scalebar is 10 µm. The representive frames in the still image are 15 seconds apart.

Schematic representation of one period of the actin filament consisting of 28 actin monomers. The monomers pair up into dimers which then twist helically with a full twist occurring every 14 dimers which is approximately 74 nm in length [10,12,13].

Optical torques acting upon a bundle of 25 filaments as a function of its orientation with respect to the major axis of the line trap. Fig. 7a represents the torque as a function of angle for a Gaussian beam of dimensions 7 µm×7 µm and Fig. 7b is for a line trap of dimensions 1 µm×45 µm. Both lasers have a total power of 75 mW.

Graphical representation of filopodia equilibrium orientations in optical traps. Fig. 8a represents filopodia of 2 µm length being fixed at one end in the Gaussian beam. A grayscale intensity profile of the beam is shown behind the filopodia. Fig. 8b shows the same filopodia in the line trap. The dashed line links the points of maximum intensity between the two profiles.